I have over 12 years of experience in the area of viral pathogenesis and immunity. My doctoral studies and postdoctoral training gave me a broad understanding of poxvirus biology, and of the biology of xenotransplantation. My more recent work, which forms the basis of the science in the current application, is focused on generation and study of human monoclonal antibodies to dengue viruses. This information is of timely importance, as it is needed for the rational design of an effective dengue virus vaccine. I am also trained, and board certified, in Internal Medicine and as an Infectious Diseases clinical specialist. My long-term career goal is to remain in academic medicine as a physician-scientist and conduct translational research in immunovirology while growing my clinical skills and knowledge of infectious diseases. By utilizing the knowledge that I gain from this proposed work, I hope to develop my career in a direction independently of my mentor and begin to work on the rational design of a hepatitis C virus vaccine. My surroundings at Vanderbilt are ideal for my proposed project and my career development. The intellectual environment could not be better, and I intend to take full advantage of this by attending a structured program of coursework. My mentor's laboratory has a tremendous amount of experience isolating and studying human monoclonal antibodies, central to my proposal and to the concept of rational vaccine design. My mentor, who is the director of the Vanderbilt Vaccine Center, also has great expertise in vaccinology, immunology, and viral pathogenesis. While at Vanderbilt and under his mentorship, my work has been rapidly evolving so as to launch my career as an independent investigator. Symptomatic dengue virus infection ranges in disease severity from an influenza-like illness to life-threatening shock. One model of the mechanism underlying severe disease proposes that weakly cross-reactive antibodies induced during a primary infection facilitate virus entry into Fc receptor-bearing cells during a subsequent secondary infection. This is thought to increase viral replication and release of cytokines and vasoactive mediators, culminating in shock. This unique process, known as antibody-dependent enhancement of infection, has significantly hindered vaccine development. There is a concern that potent neutralizing antibodies must be generated to all four dengue virus serotypes, as a vaccine that induces weakly cross-reactive, non-neutralizing antibodies may increase the likelihood of developing severe disease upon re-exposure. Much of our understanding of this process has come from studies using mouse mAbs. However, antibody responses in mice typically exhibit less complexity than those in humans. A better understanding of the humoral immune response to natural dengue virus infection in humans is sorely needed. Using a high-efficiency human hybridoma technology developed in our laboratory, it is now possible to generate human hybridomas reliably with B cells from the peripheral blood of individuals who have recovered from an infection. Employing this technology, we have generated over 250 hybridomas secreting human mAbs to dengue virus from subjects who had recovered from primary or secondary infection. The vast majority of these antibodies are broadly serotype cross-reactive, directed against either envelope or pre-membrane protein, and capable of antibody-mediated enhancement of infection. Interestingly, very rare serotype-specific, potently neutralizing antibodies, which are nearly devoid of enhancing activity, are also produced by humans in response to infection. Understanding the epitopes and activity of these naturally-occurring antibodies is critical for vaccine development, as vaccines that induce high potency neutralizing antibodies that lack enhancing activity are desirable. Ideally, the reactivity of epitopes bound by enhancing antibodies should be reduced or eliminated in candidate antigens during the rational development of a dengue vaccine, so as to discourage such dominant recognition of these antigenic features by the humoral immune response. The long-term goal is to use such molecular information in the rational design of dengue vaccines that enhance the induction of protective neutralizing antibodies and reduce the risk of development of severe disease. NARRATIVE: This mentored proposal outlines ambitious studies and career development, in which I will isolate potently neutralization human monoclonal antibodies to dengue viruses and determine the biochemical, structural, and genetic basis for their binding. In the process of determining fine details of the dengue virus neutralizing immune response, I will acquire skills in the areas of virology, immunology, genetics and recombinant antibody techniques. These skills, along with didactic coursework, will help me to develop my career, as a physician-scientist, in a direction independently of my mentor.